Targeting gene product in tumors is very important for systemic gene delivery because only a portion of genes are successfully delivered into tumors by using the most successful systemic tumor-targeted gene delivery technology. More importantly, some gene products such as IL-12 are more effective when co-localized with tumors, compared to staying in blood or other organs One simple approach to targeting gene product into tumors is to generate and use fusion genes encoding IL-12 and tumor-targeted mini-peptide. Unexpectedly, we found that none of the known tumor-targeted peptides boost IL-12-induced anti-tumor efficacy. In the current grant period, however, we discovered the novel peptide VNTANST, which boosts accumulation of IL-12 protein into tumors when a fusion gene encoding IL-12-VNTANST, was administered into muscles distant from the tumors. Using a gene therapy approach for DNA delivery and using a fusion gene product IL-12-VNTANST for tumor-targeting (tumor-targeted IL-12 gene therapy has shown great potential in inhibiting tumors in multiple tumor models. We also revealed that this tumor-targeting IL-12 (IL-12-VNTANST) binds to vimentin, a protein known as an intracellular protein and increased in metastatic tumors. An intriguing question is how the extracellular IL-12-VNTANST binds to an intracellular protein? Significantly, our preliminary study showed that vimentin is found on the cell membrane of malignant epithelial cells. Our objective in this renewal application is to determine whether membrane vimentin localization is increased in some of the metastatic tumor cells and thus has a significant role in binding to IL-12-VNTANST and boosting IL-12-anti-tumor efficacy. We will also examine how to effectively using this targeted IL-12 gene therapy for treating membrane positive metastatic tumors in various organs using tumor-targeted IL-12 gene therapy. We hypothesize that tumor-targeted IL-12- VNTANST gene therapy has a great potential for treating membrane vimentin positive metastatic tumors. The proposed study is innovative because it may definitively confirm that the membrane vimentin localization plays a key role for tumor metastasis and for boosting the proposed targeted IL-12 gene therapy. Confirmation of membrane vimentin dependence would also allow us selecting proper tumor-bearing patient in the future to perform this therapy, which is so called personalized immune therapy, an important direction of novel therapeutics and a long term of goal of our effort. PUBLIC HEALTH RELEVANCE: Tumor recurrence and metastasis after primary therapy are the major causes of death in cancer patients. Metastatic tumor-targeted therapy is lacking. In this application, we will determine how to effectively use this targeted immune therapy for treating metastatic tumors in different organs with a goal of moving this therapy in human clinics.